Research in VeterinaryScience 1991, 51, 313-316
Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro P. D. MANSELL, B. W. PARRY, Department o f Veterinary Science, University o f Melbourne, Werribee, Victoria 3030, Australia
The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 °, 20 ° and 37°C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 ° or 20°C. The concentration of yon Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.
FACTOR VIII (FVIII, antihaemophilic factor) is the component of the intrinsic cascade of blood coagulation which is deficient in individuals with haemophilia A. von Willebrand factor (vWf) is a plasma protein involved in adhesion of platelets to subendothelial collagen or to other platelets and is the deficient factor in von Willebrand's disease. Assay of FVIII and vWf is necessary for the definitive diagnosis of haemophilia A and von Willebrand's disease. In addition, the ratio of FVIII and vWf in plasma may be used to assist the detection of females heterozygous for the haemophilia A gene, because heterozygotes generally have a lower ratio of FVIII to vWf than normal females (Zimmerman et al 1971, Johnstone and Norris 1984). The assays of canine FVIII and vWf are specialised procedures and there is often a delay between collection of blood samples and their processing in a suitable laboratory. Factor VIII is considered to be one of the most labile of the coagulation factors in human plasma (Hondow et al 1982a). Human vWf is more stable in vitro than is human FVII (Eyster et al 1976, Nilsson et al 1983). In contrast, canine FVIII activity is relatively stable in vitro, both in samples stored as plasma and those stored as whole blood. Samples of canine whole blood stored at 4°C for 48 hours retained 81 per cent of the original FVIII activity (Mansell and Parry 1989). Samples stored at 20°C for 48 hours retained 97 per cent of the original FVIII activity. Benson et al (1983) found that the vWf
antigen (vWf:Ag) concentration was stable for up to nine days in plasma samples stored at 2°C, but only for shorter periods at higher temperatures. Dodds (1978) recommended that canine l~lood samples for coagulation assays should be submitted as fresh frozen plasma maintained at - 20°C or lower. Studies of the stability of canine vWf in whole blood in vitro have not been reported. Similarly, there have been no reports of the stability of the ratio of FVIII and vWf activities in canine blood samples in vitro. The purpose of the present study was to investigate the stability of FVIII activity, vWf:Ag concentration and the ratio of FVIII activity and vWf:Ag concentration (FVIII/vWf ratio) in canine blood samples in vitro. Materials and methods
Twelve adult, clinically normal, mixed breed dogs (six male, six female) were used. Blood samples were collected from a jugular vein, using a plastic syringe containing 1/10 volume of 3"8 per cent sodium citrate. Samples to be stored as plasma were centrifuged at 2000 g for 10 minutes and stored in plastic tubes. Samples to be held as whole blood were stored in plastic tubes and centrifuged just before assay. The samples of plasma and whole blood were held at 4, 20 and 37°C. The degree of haemolysis in stored whole blood samples was assessed visually and graded as nil, slight, moderate or severe. Baseline FVIII activities and vWf:Ag concentrations were determined for each dog within 30 minutes of sample collection. Two samples were assayed, one of which was used as the baseline value for the plasma samples and the other used for the baseline value of the whole blood samples. The FVIII activity and vWf'Ag concentration of stored samples were measured 24 and 48 hours after sample collection. Factor VIII activity was measured by a modified onestage activated partial thromboplastin time as previously described (Parry et al 1988). The concentration of vWf:Ag was measured by immunoelectrophoresis using anti-canine vWf antibodies,
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P. D. Mansell, B. IV. Parry TABLE 1 : Effect of storage temperature and storage time on canine factor VIII activity Storage temperature Plasma 4°C 20°C 37°C Whole blood 4°C 20°C 37°C
0
Time after sample collection (h) 24
48
9 5 . 4 4- 3 . 9 a 95.4 ± 3.9 a 95,4 ± 3.9 a
93.2 ± 5.8 a 8 8 . 5 4- 3 . 4 a 60.9 ± 6.4 b
76.8 ± 2.7 b 74.5 ± 4.2 b 1 6 . 4 4- 6 . 8 c
101 . 7 ± 5 . 1 a 101 . 7 4- 5 . 1 a 101 . 7 ± 5 . 1 a
87-1 ± 3.8 b 93-9 ± 2.5 a 86-1 ± 4.7 a
70.1 ± 4.0 c 89-5 ± 5.2 a 101-2-~ 8.5 a
Values are canine units d l - 1 mean ± SEM, n = 10 Values in the same row which have different superscripts are significantly different (P 0.05). Generally, stability was greater in plasma samples, and in samples stored at lower temperature. The stability of the ratio of FVIII activity and vWf:Ag concentration, in plasma and in whole blood (Table 3), reflected the changes in both FVIII activity and vWf:Ag concentration described above. Interaction between type of sample and temperature of storage was significant during the periods 0 to 48 (P
Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro P. D. MANSELL, B. W. PARRY, Department o f Veterinary Science, University o f Melbourne, Werribee, Victoria 3030, Australia
The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 °, 20 ° and 37°C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 ° or 20°C. The concentration of yon Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.
FACTOR VIII (FVIII, antihaemophilic factor) is the component of the intrinsic cascade of blood coagulation which is deficient in individuals with haemophilia A. von Willebrand factor (vWf) is a plasma protein involved in adhesion of platelets to subendothelial collagen or to other platelets and is the deficient factor in von Willebrand's disease. Assay of FVIII and vWf is necessary for the definitive diagnosis of haemophilia A and von Willebrand's disease. In addition, the ratio of FVIII and vWf in plasma may be used to assist the detection of females heterozygous for the haemophilia A gene, because heterozygotes generally have a lower ratio of FVIII to vWf than normal females (Zimmerman et al 1971, Johnstone and Norris 1984). The assays of canine FVIII and vWf are specialised procedures and there is often a delay between collection of blood samples and their processing in a suitable laboratory. Factor VIII is considered to be one of the most labile of the coagulation factors in human plasma (Hondow et al 1982a). Human vWf is more stable in vitro than is human FVII (Eyster et al 1976, Nilsson et al 1983). In contrast, canine FVIII activity is relatively stable in vitro, both in samples stored as plasma and those stored as whole blood. Samples of canine whole blood stored at 4°C for 48 hours retained 81 per cent of the original FVIII activity (Mansell and Parry 1989). Samples stored at 20°C for 48 hours retained 97 per cent of the original FVIII activity. Benson et al (1983) found that the vWf
antigen (vWf:Ag) concentration was stable for up to nine days in plasma samples stored at 2°C, but only for shorter periods at higher temperatures. Dodds (1978) recommended that canine l~lood samples for coagulation assays should be submitted as fresh frozen plasma maintained at - 20°C or lower. Studies of the stability of canine vWf in whole blood in vitro have not been reported. Similarly, there have been no reports of the stability of the ratio of FVIII and vWf activities in canine blood samples in vitro. The purpose of the present study was to investigate the stability of FVIII activity, vWf:Ag concentration and the ratio of FVIII activity and vWf:Ag concentration (FVIII/vWf ratio) in canine blood samples in vitro. Materials and methods
Twelve adult, clinically normal, mixed breed dogs (six male, six female) were used. Blood samples were collected from a jugular vein, using a plastic syringe containing 1/10 volume of 3"8 per cent sodium citrate. Samples to be stored as plasma were centrifuged at 2000 g for 10 minutes and stored in plastic tubes. Samples to be held as whole blood were stored in plastic tubes and centrifuged just before assay. The samples of plasma and whole blood were held at 4, 20 and 37°C. The degree of haemolysis in stored whole blood samples was assessed visually and graded as nil, slight, moderate or severe. Baseline FVIII activities and vWf:Ag concentrations were determined for each dog within 30 minutes of sample collection. Two samples were assayed, one of which was used as the baseline value for the plasma samples and the other used for the baseline value of the whole blood samples. The FVIII activity and vWf'Ag concentration of stored samples were measured 24 and 48 hours after sample collection. Factor VIII activity was measured by a modified onestage activated partial thromboplastin time as previously described (Parry et al 1988). The concentration of vWf:Ag was measured by immunoelectrophoresis using anti-canine vWf antibodies,
313
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P. D. Mansell, B. IV. Parry TABLE 1 : Effect of storage temperature and storage time on canine factor VIII activity Storage temperature Plasma 4°C 20°C 37°C Whole blood 4°C 20°C 37°C
0
Time after sample collection (h) 24
48
9 5 . 4 4- 3 . 9 a 95.4 ± 3.9 a 95,4 ± 3.9 a
93.2 ± 5.8 a 8 8 . 5 4- 3 . 4 a 60.9 ± 6.4 b
76.8 ± 2.7 b 74.5 ± 4.2 b 1 6 . 4 4- 6 . 8 c
101 . 7 ± 5 . 1 a 101 . 7 4- 5 . 1 a 101 . 7 ± 5 . 1 a
87-1 ± 3.8 b 93-9 ± 2.5 a 86-1 ± 4.7 a
70.1 ± 4.0 c 89-5 ± 5.2 a 101-2-~ 8.5 a
Values are canine units d l - 1 mean ± SEM, n = 10 Values in the same row which have different superscripts are significantly different (P 0.05). Generally, stability was greater in plasma samples, and in samples stored at lower temperature. The stability of the ratio of FVIII activity and vWf:Ag concentration, in plasma and in whole blood (Table 3), reflected the changes in both FVIII activity and vWf:Ag concentration described above. Interaction between type of sample and temperature of storage was significant during the periods 0 to 48 (P
